Apoptotic surface delivery of K+ channels

Apoptosis in cortical neurons requires efflux of cytoplasmic potassium mediated by a surge in Kv2.1 channel activity. Pharmacological blockade or molecular disruption of these channels in neurons prevents apoptotic cell death, while ectopic expression of Kv2.1 channels promotes apoptosis in non-neuronal cells. Here, we use a cysteine-containing mutant of Kv2.1 and a thiol-reactive covalent inhibitor to demonstrate that the increase in K+ current during apoptosis is due to de novo insertion of functional channels into the plasma membrane. Biotinylation experiments confirmed the delivery of additional Kv2.1 protein to the cell surface following an apoptotic stimulus. Finally, expression of botulinum neurotoxins that cleave syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) blocked upregulation of surface Kv2.1 channels in cortical neurons, suggesting that target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins support proapoptotic delivery of K+ channels. These data indicate that trafficking of Kv2.1 channels to the plasma membrane causes the apoptotic surge in K+ current.     

Subcellular segregation of two A-type K+ channel proteins in rat central neurons.

In the mammalian nervous system, K+ channels regulate diverse aspects of neuronal function and are encoded by a large set of K+ channel genes. The roles of different K+ channel proteins could be dictated by their localization to specific subcellular domains. We report that two K+ channel polypeptides, Kv1.4 and Kv4.2, which form transient (A-type) K+ channels when expressed in Xenopus oocytes, are segregated in rat central neurons. Kv1.4 protein is targeted to axons and possibly terminals, while Kv4.2 is concentrated in dendrites and somata. This differential distribution implies distinct roles for these channel proteins in vivo. Their localizations suggest that Kv1.4 and Kv4.2 may regulate synaptic transmission via presynaptic, or postsynaptic mechanisms, respectively.     

KChAP/Kvbeta1.2 interactions and their effects on cardiac Kv channel expression

KChAP and voltage-dependent K+ (Kv) beta-subunits are two different types of cytoplasmic proteins that interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and Kv4.3 channels. It also binds to Kv1.x channels but, with the exception of Kv1.3, does not increase Kv1.x currents. Kvbeta-subunits are assembled with Kv1.x channels; they exhibit "chaperone-like" behavior and change gating properties. In addition, KChAP and Kvbeta-subunits interact with each other. Here we examine the consequences of this interaction on Kv currents in Xenopus oocytes injected with different combinations of cRNAs, including Kvbeta1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found that KChAP attenuated the depression of Kv1.5 currents produced by Kvbeta1.2, and Kvbeta1.2 eliminated the increase of Kv2.1 and Kv4.3 currents produced by KChAP. Both KChAP and Kvbeta1.2 are expressed in cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward currents and Kv4.3 and Kv1.4 generate transient outward currents. Because they interact, either KChAP or Kvbeta1.2 may alter both sustained and transient cardiac Kv currents. The interaction of these two different classes of modulatory proteins may constitute a novel mechanism for regulating cardiac K+ currents.     

Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells

Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to K+ efflux; blockade of plasmalemmal K+ channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated K+ (Kv) channels. A human bcl-2 gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents (I(Kv)). In contrast, the apoptosis inducer staurosporine (ST) enhanced I(Kv). In bcl-2-infected cells, however, the ST-induced increase in I(Kv) was completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (-bcl-2). Blockade of Kv channels in control cells (-bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in I(Kv) and apoptosis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of I(Kv) and downregulated the mRNA expression of the pore-forming Kv channel alpha-subunits (Kv1.1, Kv1.5, and Kv2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells.     

Molecular diversity and function of voltage-gated (Kv) potassium channels in epithelial cells

Voltage-gated K+ channels belonging to Kv1-9 subfamilies are widely expressed in excitable cells where they play an essential role in membrane hyperpolarization during an action potential and in the propagation of action potentials along the plasma membrane. Early patch clamp studies on epithelial cells revealed the presence of K+ currents with biophysical and pharmacologic properties characteristic of Kv channels expressed in excitable cells. More recently, molecular approaches including PCR and the availability of more selective antibodies directed against Kv alpha and auxiliary subunits, have demonstrated that epithelial cells from various organ systems, express a remarkable diversity Kv channel subunits. Unlike neurons and myocytes however, epithelial cells do not typically generate action potentials or exhibit dynamic changes in membrane potential necessary for activation of Kv alpha subunits. Moreover, the fact that many Kv channels expressed in epithelial cells exhibit inactivation suggest that their activities are relatively transient, making it difficult to ascribe a functional role for these channels in transepithelial electrolyte or nutrient transport. Other proposed functions have included (i) cell migration and wound healing, (ii) cell proliferation and cancer, (iii) apoptosis and (iv) O2 sensing. Certain Kv channels, particularly Kv1 and Kv2 subfamily members, have been shown to be involved in the proliferation of prostate, colon, lung and breast carcinomas. In some instances, a significant increase in Kv channel expression has been correlated with tumorogenesis suggesting the possibility of using these proteins as markers for transformation and perhaps reducing the rate of tumor growth by selectively inhibiting their functional activity.     

Voltage-dependent Ca(2+) channel subunit expression and immunolocalization in mouse spermatogenic cells and sperm

Though voltage-dependent Ca(2+) channels contribute to the orchestration of sperm differentiation and function, many questions remain concerning their molecular architecture. This study shows that alpha(1A) and alpha(1C) Ca(2+) channel pore-forming subunits are expressed in spermatogenic cells. In addition, it provides what is to our knowledge the first evidence for the presence of the Ca(2+) channel beta auxiliary subunits in spermatogenic cells and sperm. Using RT-PCR we demonstrated the expression of all four known genes encoding the beta subunits in spermatogenic cells. Specific antibodies detected three of these proteins in spermatogenic cells and sperm. In spermatogenic cells both alpha(1) and beta subunits are diffusely distributed throughout the cytoplasm while in sperm they appear to be regionally localized.     

Anorexic effect of K+ channel blockade in mesenteric arterial smooth muscle and intestinal epithelial cells.

Activity of voltage-gated K+ (Kv) channels controls membrane potential (E(m)). Membrane depolarization due to blockade of K+ channels in mesenteric artery smooth muscle cells (MASMC) should increase cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and cause vasoconstriction, which may subsequently reduce the mesenteric blood flow and inhibit the transportation of absorbed nutrients to the liver and adipose tissue. In this study, we characterized and compared the electrophysiological properties and molecular identities of Kv channels and examined the role of Kv channel function in regulating E(m) in MASMC and intestinal epithelial cells (IEC). MASMC and IEC functionally expressed multiple Kv channel alpha- and beta-subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv4.3, and Kv9.3, as well as Kvbeta1.1, Kvbeta2.1, and Kvbeta3), but only MASMC expressed voltage-dependent Ca2+ channels. The current density and the activation and inactivation kinetics of whole cell Kv currents were similar in MASMC and IEC. Extracellular application of 4-aminopyridine (4-AP), a Kv-channel blocker, reduced whole cell Kv currents and caused E(m) depolarization in both MASMC and IEC. The 4-AP-induced E(m) depolarization increased [Ca2+]cyt in MASMC and caused mesenteric vasoconstriction. Furthermore, ingestion of 4-AP significantly reduced the weight gain in rats. These results suggest that MASMC and IEC express multiple Kv channel alpha- and beta-subunits. The function of these Kv channels plays an important role in controlling E(m). The membrane depolarization-mediated increase in [Ca2+]cyt in MASMC and mesenteric vasoconstriction may inhibit transportation of absorbed nutrients via mesenteric circulation and limit weight gain.     

Kvbeta1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to kv4.2 but not to Shaker channels.

Voltage-gated K+ (KV) channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic modulatory beta subunits. The differential expression and association of alpha and beta subunits seems to contribute significantly to the complexity and heterogeneity of KV channels in excitable cells, and their functional expression in heterologous systems provides a tool to study their regulation at a molecular level. Here, we have studied the effects of Kvbeta1.2 coexpression on the properties of Shaker and Kv4.2 KV channel alpha subunits, which encode rapidly inactivating A-type K+ currents, in transfected HEK293 cells. We found that Kvbeta1.2 functionally associates with these two alpha subunits, as well as with the endogenous KV channels of HEK293 cells, to modulate different properties of the heteromultimers. Kvbeta1.2 accelerates the rate of inactivation of the Shaker currents, as previously described, increases significantly the amplitude of the endogenous currents, and confers sensitivity to redox modulation and hypoxia to Kv4.2 channels. Upon association with Kvbeta1.2, Kv4.2 can be modified by DTT (1,4 dithiothreitol) and DTDP (2,2'-dithiodipyridine), which also modulate the low pO2 response of the Kv4.2+beta channels. However, the physiological reducing agent GSH (reduced glutathione) did not mimic the effects of DTT. Finally, hypoxic inhibition of Kv4.2+beta currents can be reverted by 70% in the presence of carbon monoxide and remains in cell-free patches, suggesting the presence of a hemoproteic O2 sensor in HEK293 cells and a membrane-delimited mechanism at the origin of hypoxic responses. We conclude that beta subunits can modulate different properties upon association with different KV channel subfamilies; of potential relevance to understanding the molecular basis of low pO2 sensitivity in native tissues is the here described acquisition of the ability of Kv4. 2+beta channels to respond to hypoxia.     

Differential expression of K+ channel mRNAs in the rat brain and down-regulation in the hippocampus following seizures.

K+ channels are major determinants of membrane excitability. Differences in neuronal excitability within the nervous system may arise from differential expression of K+ channel genes, regulated spatially in a cell type-specific manner, or temporally in response to neuronal activity. We have compared the distribution of mRNAs of three K+ channel genes, Kv1.1, Kv1.2, and Kv4.2 in rat brain, and examined activity-dependent changes following treatment with the convulsant drug pentylenetetrazole. Both regional and cell type-specific differences of K+ channel gene expression were found. In addition, seizure activity caused a reduction of Kv1.2 and Kv4.2 mRNAs in the dentate granule cells of the hippocampus, raising the possibility that K+ channel gene regulation may play a role in long-term neuronal plasticity.     

Solid support membranes for ion channel arrays and sensors: application to rapid screening of pharmacological compounds

The use of solid supported membranes (SSM) was investigated for reconstitution of ion channels and for potential application to screen pharmacological reagents affecting ion channel function. The voltage-gated Kv1.5 K+ channel was reconstituted on an SSM and a current was measured. This current was dependent on the presence of K+, but not Na+, indicating that the Kv1.5 K+ channel maintained cation specificity when reconstituted on SSM. Two pharmacological reagents applied to Kv1.5 K+ channels reconstituted on SSM had similar inhibitory effects as those measured using Kv1.5 in biological membranes. SSM-mounted ion channels were stable enough to be washed with buffer solution and reused many times, allowing solution exchange essential for pharmacological drug screening.     

Constitutive activation of delayed-rectifier potassium channels by a src family tyrosine kinase in Schwann cells.

In the nervous system, Src family tyrosine kinases are thought to be involved in cell growth, migration, differentiation, apoptosis, as well as in myelination and synaptic plasticity. Emerging evidence indicates that K+ channels are crucial targets of Src tyrosine kinases. However, most of the data accumulated so far refer to heterologous expression, and native K+-channel substrates of Src or Fyn in neurons and glia remain to be elucidated. The present study shows that a Src family tyrosine kinase constitutively activates delayed-rectifier K+ channels (IK) in mouse Schwann cells (SCs). IK currents are markedly downregulated upon exposure of cells to the tyrosine kinase inhibitors herbimycin A and genistein, while a potent upregulation of IK is observed when recombinant Fyn kinase is introduced through the patch pipette. The Kv1.5 and Kv2.1 K+-channel alpha subunits are constitutively tyrosine phosphorylated and physically associate with Fyn both in cultured SCs and in the sciatic nerve in vivo. Kv2.1- channel subunits are found to interact with the Fyn SH2 domain. Inhibition of Schwann cell proliferation by herbimycin A and by K+-channel blockers suggests that the functional linkage between Src tyrosine kinases and IK channels could be important for Schwann cell proliferation and the onset of myelination.     

Kv8.1, a new neuronal potassium channel subunit with specific inhibitory properties towards Shab and Shaw channels.

Outward rectifier K+ channels have a characteristic structure with six transmembrane segments and one pore region. A new member of this family of transmembrane proteins has been cloned and called Kv8.1. Kv8.1 is essentially present in the brain where it is located mainly in layers II, IV and VI of the cerebral cortex, in hippocampus, in CA1-CA4 pyramidal cell layer as well in granule cells of the dentate gyrus, in the granule cell layer and in the Purkinje cell layer of the cerebellum. The Kv8.1 gene is in the 8q22.3-8q24.1 region of the human genome. Although Kv8.1 has the hallmarks of functional subunits of outward rectifier K+ channels, injection of its cRNA in Xenopus oocytes does not produce K+ currents. However Kv8.1 abolishes the functional expression of members of the Kv2 and Kv3 subfamilies, suggesting that the functional role of Kv8.1 might be to inhibit the function of a particular class of outward rectifier K+ channel types. Immunoprecipitation studies have demonstrated that inhibition occurs by formation of heteropolymeric channels, and results obtained with Kv8.1 chimeras have indicated that association of Kv8.1 with other types of subunits is via its N-terminal domain.     

Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five- fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage- gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.